Earlier work supported by this grant developed the chemistry and structural physics of protein hydrogen exchange (HX) and showed, given this fundamental information, how HX methods can be used to measure protein structure, stability, dynamics, binding, structure change, and folding. These studies led to the discovery of the previously unsuspected foldon dimension of protein structure. Results in our lab and others now suggest that proteins fold by putting cooperative units of the native structure (foldons) into place in a predetermined sequential manner, with prior structure acting as a template to support the formation and docking of incoming native-like foldon units. It also appears that proteins can use their foldons to control other functions including the rate of ligand binding and release and the stability and kinetics of structure change processes. This application proposes to further investigate the implications of the newly discovered foldon dimension of protein structure for protein folding and function. Aims are as follows. (1) Continue work on foldons and folding in the major Staphylococcal Nuclease model. (2) Investigate the folding behavior of larger proteins, using maltose binding protein (41 kDa) as an initial model. (3) Study the role of transient partially unfolded states in other protein functions, with initial work directed at the Mad2 mitotic checkpoint protein. These Aims will exploit the same powerful suite of integrated hydrogen exchange methods that were developed and used successfully in earlier studies together with newly developed fast 3D NMR and enhanced HX/mass spectrometry methods. PUBLIC HEALTH RELEVANCE: Hydrogen exchange knowledge and methods developed in our earlier work have led to the discovery of a whole new dimension of protein structure and function based on cooperative foldon units. Foldons appear to provide the key to the protein folding and misfolding problem and to underlie some other protein functions as well. In order to understand the fundamental roles of protein molecules in health and disease, the experimentation proposed here aims to explore the implications of the newly discovered foldon dimension of protein structure for protein folding and misfolding and for protein function more broadly.